Production of IFNa is one of the earliest host immune responses after viral infection. The fundamental importance of this Type I interferon, lies in its ability to induce downstream activation of both innate and adaptive immune cells via direct and indirect mechanisms. Because they promote potent anti-viral activity, Type I IFNs have been used as both a monotherapy and in combination with other small molecules for enhanced efficacy in the treatment of chronic viral infections, such as Hepatitis C. Despite the fact that only a portion of patients respond to treatment, and that adverse side-effects from long term dosing regimens are a significant barrier, IFNs remain the best standard of care. Thus, it is the overall goal of this project to develop, in parallel, two classes of IFNa enhancers, one protein and the other small molecule, that may be used alone or in combination with other therapeutics for the broad-spectrum treatment of viral infections. First, in an effort to improve functional responses to IFNs and reduce their toxicity, the Garcia group has developed an approach to re-engineer and expand the menu of existing IFNs with recombinant proteins that exhibit superior therapeutic properties. This method, known as in vitro evolution, has been previously and successfully employed by the Garcia group to engineer cytokines with unique structure-activity relationships and that have proven to be more efficacious than the natural cytokine. Second, the Khosia group will pursue chemical biological studies on a recently discovered polyketide natural product, A-74528, that has been found to enhance the antirviral effects of IFNa. The Khosia group will also engage in production and biochemical and structural analysis of A-74528 analogs. In Aim 1 we will engineer and improve IFN antiviral activities using in vitro evolution to create IFNs with altered affinity or dimerization geometries with the receptor complex. In Aim 2 we will conduct biophysical studies of candidate IFNs to determine their mechanisms of action, as monotherapies or in combination with other therapeutics. In Aim 3 we will investigate the inhibitory mechanism and pharmacological properties of A- 74528 through kinetic and dose-response analyses. In Aim 4 we will design and synthesize analogs of A- 74528 to define structure activity relationships. This unique interfacing of protein and small molecule medicinal chemistry will hopefully yield highly granular, predictive structure-activity metrics linking the molecular and mechanistic parameters of IFN action to antiviral function.